Sasaki Kohei, Hayashi Kyoko, Lee Jung-Bum, Kurosaki Fumiya, Hayashi Toshimitsu
Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama, Toyama, 930-0194, Japan.
Research Institute of Life and Health Sciences, Chubu University, Kasugai, Aichi, 487-8501, Japan.
PLoS One. 2015 Mar 20;10(3):e0121205. doi: 10.1371/journal.pone.0121205. eCollection 2015.
It is generally accepted that live attenuated influenza vaccine (LAIV) has the potential for use as a vaccination against flu. In this study, we demonstrated the nature of an influenza A virus (IAV) mutant induced by treating the IAV with a stable furan derivative, (1R,2R)-1-(5'-methylfur-3'-yl)propane-1,2,3-triol (MFPT), which had been isolated from Streptomyces sp. strain FV60 with the objective of it being an LAIV candidate. The resulting MFPT-resistant (MFPTr) IAVs possessed attenuated pathogenicity in vitro and in vivo when compared with that of the parent virus (H1N1 subtype, NWS strain). Sequencing analysis revealed that a novel mutation, C490U in ns gene (P164S in NS1), was detected in all MFPTr virus clones tested. Therefore, NS1 might be a main target of MFPT, and it was suggested that the P164S mutation contributed to the attenuated pathogenicity of the mutants. Although the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is one of the targets of NS1, the MFPTr virus suppressed the phosphorylation of Akt when compared with the wild-type (WT) virus. It was suggested that this might lead to the subsequent inhibition of the cleavage of PARP-1 and caspase-3, which is important for the progression of apoptosis. At the same time, nucleoprotein (NP) was found to be retained in the nuclei in MFPTr virus-infected cells while nuclear export of NP was detected in WT virus-infected cells. In addition, the expression levels of interferon-β transcripts were significantly decreased in MFPTr virus-infected cells. From these results it can be shown that the mutation, NS1P164S, might be one of the key residues to control NS1 function concerning the induction of apoptosis. In conclusion, MFPT induced favorable mutation in the ns gene for the attenuation of IAV, and therefore might provide the novel methodology for preparing LAIVs.
普遍认为，减毒活流感疫苗（LAIV）有作为流感疫苗使用的潜力。在本研究中，我们展示了用一种稳定的呋喃衍生物（1R,2R）-1-（5'-甲基呋喃-3'-基）丙烷-1,2,3-三醇（MFPT）处理甲型流感病毒（IAV）所诱导的IAV突变体的性质，该衍生物是从链霉菌属菌株FV60中分离出来的，目的是将其作为LAIV候选物。与亲本病毒（H1N1亚型，NWS株）相比，所得的耐MFPT（MFPTr）IAV在体外和体内具有减弱的致病性。测序分析表明，在所有测试的MFPTr病毒克隆中都检测到一个新的突变，即ns基因中的C490U（NS1中的P164S）。因此，NS1可能是MFPT的主要靶点，并且表明P164S突变导致了突变体致病性的减弱。虽然磷脂酰肌醇3-激酶（PI3K）/Akt信号通路是NS1的靶点之一，但与野生型（WT）病毒相比，MFPTr病毒抑制了Akt的磷酸化。有人认为，这可能导致随后对PARP-1和caspase-3裂解的抑制，这对细胞凋亡的进展很重要。同时，发现核蛋白（NP）在MFPTr病毒感染的细胞中保留在细胞核中，而在WT病毒感染的细胞中检测到NP的核输出。此外，在MFPTr病毒感染的细胞中，干扰素-β转录本的表达水平显著降低。从这些结果可以看出，NS1P164S突变可能是控制NS1与细胞凋亡诱导相关功能的关键残基之一。总之，MFPT诱导了IAV减毒的ns基因中的有利突变，因此可能为制备LAIV提供新的方法。
